The biochip is a representative example of novel technology combining material technology such as nanotechnology (NT), biotechnology (BT) which is contents and applied field of the material technology and information technology (IT) to analyze a large amount of results. The biochip is formed by high-density micro-arraying of various kinds of biomaterials on a unit area of a surface of a solid support. The biochip technologies include a technology to immobilize biomaterials, a technology to make the chip surface biocompatible, microarray technology of biomaterials, an assay technology to perform various biological reactions on a produced chip, a technology to detect reaction results, a protein engineering to produce biomaterials to be immobilized and genetic recombination technology.
A protein chip representative of the biochips is formed by intensive micro-arraying of various proteins on a unit area of the surface of a solid support. By using the protein chip, it is possible to conduct an experiment for multiple purposes, such as diagnosis of diseases, high throughput screening (HTS), enzyme activity test and the like, with a small amount of samples.
There have been attempts to produce the protein chip by employing the same principles and technical factors as in the production of DNA chips, which have been already developed and commonly used. Generally, most of the commonly used DNA chips are produced by immobilizing DNA on a glass substrate, the surface of which has been pretreated with a coating material. When the protein chip is fabricated according to a method similar to that used in the production of DNA chips, that is, when the protein chip is fabricated by immobilizing proteins on a glass substrate whose surface has been pretreated with a coating material, various problems are likely to occur due to the difference in physical and chemical properties between the target proteins to be immobilized.
Previous protein chip was produced by immobilizing proteins on a surface-treated glass substrate and used to perform a simple binding assay. The performance of the protein chip was determined by the activity of the immobilized protein and it was hard to work successfully (MacBeath and Schreiber, Science 289:1760, 2000). Such problems are caused by the denaturation, inactivation and degradation of proteins resulting from the difference in the inherent physical and chemical properties of proteins.
In order to solve these problems, researches and studies have been conducted on surface treatment technology for immobilizing proteins suitable for protein characteristics which are distinguished from those of DNA and on materials for immobilizing protein. Such research and studies are focused on a method for performing immobilization on the surface of a protein chip while maintaining the activity of the protein. Examples thereof include a hydrogel-coated slide (PerkinElmer), Versalinx chip (Prolinx), PDC chip which is a biochip commercially available from Zyomyx, etc.
In particular, the hydrogel-coated slide is a technology using a 3-dimensional polyacrylamide gel, in which a Swiss glass with an optically level, silane treated surface is used as a base material and a surface-modified acrylamide polymer is applied thereon to improve the binding force and structural stability of a protein. Herein, the protein is immobilized by a covalent bond with a functional group of polyacrylamide gel. Also, the Versalinx chip of Prolinx comprises a self-assembly monolayer of biotin-conjugated poly(L-lysine)-g-poly(ethylene glycol) formed on a TiO3, in which a protein is immobilized on the self-assembly monolayered surface, whereby the activity of the protein can be improved. These methods form a 3-dimensional micro-structure and covalently immobilize proteins on a modified surface so as to maintain the activities of proteins within spots. In addition to these methods, it is also possible to make micro-well type of chips through microprocessing so as to produce solution-state chips.
Meanwhile, a sol-gel process is a technology which has been used to make a micro-structure by microprocessing, and in particular, it is a technology comprised of forming a binding net by a mild process and immobilizing biomolecules within the binding net by methods other than a covalent bond, instead of chemically attaching biomolecules to an inorganic material (Gill, I. and Ballesteros, A., Trends Biotechnol., 18:282, 2000). Biomolecules including enzymes are immobilized on a mass sol-gel matrix for use in the production of biocatalysts or biosensors (Reetz et al., Adv. Mater., 9:943, 1997). Specially, the sol-gel matrix is also used in the detection of optical color development due to its transparency and optical property (Edminston et al., J. Coll. Interf. Sci., 163:395, 1994). Also, biomolecules are known to be not only chemically but also thermally stabilized when they are immobilized on a sol-gel matrix (Dave et al., Anal. Chem., 66:1120, 1994).
In case of the biosensor, the sol-gel reaction is used as a method for patterning by forming a micro structure on a solid support as well as for simple immobilization.
Herein, the patterning method includes shaping a liquid-state sol using a mold by fluid dynamics, gelatinizing the shaped material and separating the mold to form a pattern. For example, a technology designated as micro-moduling in-capillaries (MIMIC) technology is for patterning mesoscopic silica (Kim et al., J. Ferment. Bioeng. 82:239, 1995; Marzolin et al., Adv. Mater. 10:571, 1998; Schuller et al., Appl. Optics 38:5799, 1999). This technology can be used in basic patterning of micro-fluid engineering.
However, since the activity of protein can be affected by various factors such as pH, it is important to set conditions for the maintenance of the activity by adding protein from its sol state in the sol-gel process. For this purpose, technologies for patterning a protein by previously mixing the protein with a sol using various mild conditions such as neutral pH (Kim et al., Biotechnol. Bioeng. 73:331 to 337, 2001) are proposed, but there are problems in that the sol-gel process rapidly progresses at neutral pH to form a gel and cracks may occur or the gel turns opaque, according to the choice of additives.
To overcome the above-described problems, the present inventors previously developed a biochip using a sol-gel reaction (Korean Patent Laid-Open Publication No. 10-2004-0024510). Specifically, in the prior art, because there was no technology by which a sol-gel matrix containing biomaterials, including proteins, could be immobilized on a chip substrate in the form of spots, there was no biochip in which a sol-gel matrix having biomaterials immobilized thereon was integrated in the form of spots. In the prior patent, a technology of treating the surface of a chip substrate with a special coating material was developed, and thus a biochip employing a sol-gel reaction on the chip substrate could be prepared for the first time. By the technology of treating the chip substrate surface according to the prior patent, a sol mixture containing biomaterials can be integrated on the chip substrate in the form of spots, a sol-gel reaction for gelling the sol mixture can occur on the chip substrate, and sol-gel matrixes can be immobilized on the chip substrate. In particular, in the prior patent, unlike the prior biochips having biomaterials immobilized on the chip substrate surface by covalent bonds, a biochip comprising biomaterials entrapped and encapsulated in the pores of gel-type spots, which are integrated and immobilized on a chip substrate, was developed.
However, in the case of this biochip, in order to immobilize the biomaterial-containing gel spots on the substrate, the surface of the substrate should be treated with a coating agent such as polyvinyl acetate, and the sol mixture should be spotted on the surface-treated substrate. Thus, this biochip is disadvantageous in terms of cost or mass production and has a limitation in that it cannot be applied to biochips having a surface or shape which cannot be coated.
Accordingly, the present inventors have made many efforts to find a material composition for use in sol-gel encapsulation, which can strongly bind to a substrate which was not subjected to surface treatment essential for the commercial use of biochips. As a result, the present inventors have designed an efficient method for screening a sol composition and found a sol composition, which can strongly bind to a substrate surface and thus can withstand intense washing during the analysis process of biochips and, at the same time, can provide good assay results, thereby completing the present invention.